Bacteria are polarized and they exploit this property for a wide array of cellular processes ranging from signal transduction to cell cycle progression and virulence. Our goal is to understand, at the molecular level, how cell polarity is inherently established and maintained, and how the localization of protein and protein complexes at the cell poles impacts and regulates cell function. Our bacterial model of choice is the genetically tractable dimorphic bacterium Caulobacter crescentus, for which morphological and molecular manifestations of cell polarity have been well documented. In C. crescentus, each division is asymmetric, yielding daughter cells of different sizes, morphologies and fates. Chromosome segregation and cell division depend on the polarization of proteins and protein complexes. Furthermore, external organelles such as the flagellum, pili and stalk form at a specific cell pole during discrete periods of the cell cycle. The temporal and spatial regulation of polar morphogenesis and cell cycle progression is achieved by an intricate signal transduction network. A major component of this network is the CckA pathway whose components display polar localization at distinct stages of the cell cycle. The physiological function of this spatio-temporal regulation is not well understood. Thus, our first objective will be to determine how the localization and temporal regulation of the CckA pathway components affect protein function and the flow of phosphoryl groups in the pathway. This will be achieved using quantitative fluorescence microscopy, mutagenesis and phosphorylation assays. Our second objective will be to elucidate how polar protein localization is achieved. The identification of the polarity factors PopZ and TipN, which broadly affect the polar localization of proteins and protein complexes involved in different cellular functions, suggests that C. crescentus forms organizing centers at the poles to mediate, and perhaps coordinate, multiple polarized functions. As an entry point, we will study the polarizing function of PopZ and the mechanisms by which PopZ accumulates at the poles by performing microscopy experiments (in various genetic and conditional backgrounds) and biochemical assays in vitro and in vivo. Our third objective will be to uncover the symmetry-breaking mechanisms that underlie asymmetric division and the cell's ability to distinguish between the two cell poles. Since TipN affects pole identity and the polarization of cell division, the function of TipN and its possible connection with the actin-like MreB cytoskeleton will be investigated using mutagenesis, quantitative fluorescence microscopy and biochemical assays. PUBLIC HEALTH RELEVANCE Bacteria rely on cell polarization for a wide variety of processes, from essential cell cycle events to important aspects of pathogenesis. This project will provide insights into the molecular mechanisms by which bacteria can achieve and maintain cell polarization, which may lead to new, broad spectrum means to control bacterial cell growth.